Human parvovirus B19 (B19V) infection causes hydrops fetalis in pregnant women during the second- trimester. It also causes severe hematological diseases, including transient aplastic crisis in patients with a high turn-over rate f red blood cells; and chronic anemia in immunodeficient and immunocompromised patients which, in some cases, can be fatal. At present, no specific antiviral drugs or vaccines (that prevent B19V infection in high-risk groups) are available. B19V replication is highly restricted to human erythroid progenitor cells (EPCs) in the bone marrow and fetal liver. B19V infection-mediated hydrops fetalis and hematological disorders mainly result from the direct killing of the EPCs in which B19V replicates. Among DNA viruses, B19V has a unique feature in the processing of its precursor mRNA (pre-mRNA) in that all the viral mRNAs are alternatively processed from a single pre-mRNA. Alternative splicing of the B19V pre-mRNA, which is controlled by multiple splicing enhancers, plays a key role in regulating alternative polyadenylation of B19V mRNAs, and generates abundant small viral mRNAs for encoding two small non- structural viral proteins, i.e. 7.5-kDa and 11-kDa. The 11-kDa protein interacts with Grb2, a protein that links the signals mediated by the erythropoietin receptor to the Ras/MEK/ERK pathway in EPCs, and plays an important role in viral DNA replication. Importantly, B19V infection induces a DNA damage response (DDR). Activation of ATR and DNA-PKcs facilitates viral DNA replication. B19V does not use the host replication machinery to replicate its ssDNA genome; rather, it appears to induce a DDR and subsequently co-opts the host DNA repair mechanism to facilitate its own replication. Furthermore, B19V replication in EPCs is markedly increased under hypoxic conditions and is mediated via the down-regulation of MEK/ERK signaling. Over the past few years, we have established two experimental cell systems that remove two critical barriers to the study of B19V replication: an efficient system of productive B19V infection involving the ex vivo- expansion of EPCs under hypoxic conditions (which mimic the microenvironment of EPCs in human bone marrow and fetal liver), and a reverse genetics approach that involves transfection of the B19V dsDNA-form genome into UT7/Epo-S1 cell line cells cultured under hypoxic conditions. Using these two systems, as well as a newly-established in vitro assay to measure viral DNA replication, we will: i) determine the mechanisms underlying the alternative processing of B19V pre-mRNA; ii) elucidate the mechanisms by which the 11-kDa protein subverts MEK/ERK signaling to promote B19V replication; and iii) determine the mechanisms underlying DDR-facilitated B19V DNA replication. Our long-term goal is to identify the key molecular mechanisms underlying the alterative processing of the single promoter-transcribed parvoviral pre-mRNA, as well as parvovirus DNA replication, in a physiologically- relevant setting, i.e., EPCs ex vivo-expanded under hypoxic conditions for B19V in this application.